Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C-15-C-19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. Recently, two universal but mutually exclusive hydrocarbon production pathways in Cyanobacteria were discovered. In this study, we constructed a deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942. The mutant was incapable to produce alkanes and exhibited normal growth phenotype at low salinity. But, the mutant became salt sensitive. Overexpression of alkane synthesis genes from halotolerant Aphanothece halophytica in Synechococcus PCC7942 restored the growth defect. The alkane synthesis gene from halotolerant cyanobacterium A.halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results indicate the requirement of alkanes for salt tolerance, and the alkane synthesis genes from A.halophytica could be a promising candidate for future biofuel application.

The effects of long-term NaCl and KCl treatment on plant growth and betaine accumulation were investigated in sugar beet. Leaf fresh weight of the plants treated with 300 mM KCl and NaCl for 15 days were declined when compared with the control plants. Photosynthetic activity, chlorophyll content and magnesium content were decreased in the plants treated with 300 mM KCl, whereas these values were essentially maintained in the control and 300 mM NaCl treated plants. K+ content in 300 mM KCl treated plants were significantly higher than the Na+ content in the 300 mM NaCl treated plants. These effects were more severe in developing leaves than the mature leaves. Betaine and choline monooxygenase (CMO) accumulation levels were increased in plants exposed to 300 mM KCl and NaCl treatment, with a higher increase in NaCl treated plants. The betaine/Na+ ratio increased during the 300 mM NaCl treatment but remained constant during the KCl treatment. Results indicate the presence of a better adaptive system to high NaCl than KCl in developing leaves of sugar beet.

To survive under halophilic environments, halophilic microorganisms must have developed the special systems such as synthesis of osmoprotectant and sunscreen molecules. Osmoprotectants are small molecules that act as osmolytes and help organisms survive under extreme saline conditions. Examples of compatible solutes include betaines, amino acids, dimethylsulfoniopropionate, and sugars. These molecules accumulate in cells and balance the osmotic difference between the cell's surroundings and the cytosol. Compatible solutes have also been shown to play a protective role by maintaining enzyme activity under abiotic stress conditions. Their specific action is unknown but is thought that they are preferentially excluded from the proteins interface due to their propensity to form water structures. Here, we summarize recent progress on the research of osmoprotectant and sunscreen molecules in halophilic algae/cyanobacteria. Their possible biotechnological application in the field of green energy, biomedical research, and various biochemical industries were described.

Salt-affected soil is one of the most important abiotic stresses, leading to reduce rice productivity in many regions of the world. The objective of this investigation was to determine the Na + , soluble sugar and starch contents and expression of some starch-related genes in two genotypes, Pokkali (salt tolerant) and IR29 (salt sensitive), grown under 200 mM NaCl. Three-week-old rice seedlings cvs. Pokkali and IR29 were treated with 0 or 200 mM NaCl subsequently Na + , starch content, soluble sugar and growth characters were evaluated. The Na + concentration in salt-stressed seedlings cv. IR29 was reached following leaf sheath>leaf blade>root tissues. Na + accumulation in leaf blade and leaf sheath was positively related to soluble sugar enrichment (r 2 >0.68). In starch biosynthesis, OsAGPS2b mRNA expression in leaf blade of rice seedlings cv. Pokkali was up-regulated for 66%, when exposed to 200 mM NaCl for 48h, leading to starch accumulation. Soluble starch content in salt-stressed seedlings was peaked to 68.84 g g -1 FW in the leaf blade of cv. Pokkali and 165.83 g g -1 FW in leaf sheath of cv. IR29 which was confirmed by iodine dye staining. In cv. Pokkali, soluble starch in the leaf blade of salt-stressed seedlings was enhanced and correlated with Na + gathering CoroNa green emission. Growth performances of indica rice cv. IR29 seedlings were significantly declined when subjected to salt stress for 4 d. Na + absorption by root tissue was greater in IR29 than that in Pokkali. The starch concentration in salt-stressed seedlings of cv. Pokkali was the maximum to 68.8 g g -1 FW in the leaf blade, and it related to up-regulated levels of OsAGPS2b mRNA and OsGPL1. The study concludes that the regulation of carbohydrate metabolism in salt tolerant cultivar of rice may play a key role as a major salt defense mechanism when seedlings subjected to 200 mM NaCl.

Glycinebetaine (GB) is an important compatible solute for salinity tolerance in many plants. In this study, we analyzed the enzymatic activity and the expression level of betaine aldehyde dehydrogenase (BADH), an important enzyme that catalyzes the last step in the GB synthesis in Leymus chinensis , a GB-hyperaccumulating graminaceous halophyte, and compared with those of barley, a graminaceous glycophyte. We have isolated cDNAs for two BADH genes, LcBADH1 and LcBADH2 . LcBADH1 has a putative peroxisomal signal peptide (PTS1) at its C-terminus, while LcBADH2 does not have any typical signal peptide. Using immunofluorescent labeling, we showed that BADH proteins were localized to the cytosol and dot-shaped organelles in the mesophyll and bundle sheath cells of L.chinensis leaves. The affinity of recombinant LcBADH2 for betaine aldehyde was comparable to other plant BADHs, whereas recombinant LcBADH1 showed extremely low affinity for betaine aldehyde, indicating that LcBADH2 plays a major role in GB synthesis in L. chinensis . In addition, the recombinant LcBADH2 protein was tolerant to NaCl whereas LcBADH1 wasn’t. The kinetics, subcellular and tissue localization of BADH proteins were comparable between L. chinensis and barley. The activity and expression level of BADH proteins were higher in L. chinensis compared with barley under both normal and salinized conditions, which may be related to the significant difference in the amount of GB accumulation between two plants.

Beta vulgaris is a glycine betaine-accumulating dicotyledonous plant of the Chenopodiaceae family. In plants, glycine betaine is synthesized by two step oxidation of choline in chloroplasts. The first step converts choline to betaine aldehyde, which is the rate-limiting step and catalyzed by choline mono-oxygenase. The second step, conversion of betaine aldehyde to betaine, is catalyzed by betaine aldehyde dehydrogenase. Due to one of the two main sources of sucrose, sugar beet has high economic value. In sugar beet, accumulation levels of betaine are high in leaves and roots including tap roots. Sugar beet is a halophytic plant that can grow under relatively high salinity conditions concomitant with the accumulation of betaine. Recent studies suggest the importance of precursor supply for the large accumulation of betaine. In this chapter, the regulation of biosynthetic genes for betaine synthesis, precursor supply of choline and transport of betaine are discussed. Metabolic engineering and application of betaine from sugar beet are also described.

Barley (Hordeum vulgare L.) plants accumulate glycine betaine (GB), a major compatible solute, in response to salt stress. In barley, GB is produced by a two-step oxidation of choline in a cooperative way in the cytosol and peroxisomes. In this study, we investigated the localization of two GB biosynthetic enzymes, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), in the tissues of barley plants (cv. Haruna-nijyo) grown under normal and saline conditions. Three-week-old barley plants grown hydroponically were treated with a hydroponic culture solution containing 200 mM NaCl for 72 h. Salt treatment resulted in increased expression of CMO and BADH proteins mainly in the leaves of barley but not in the roots. The expression of CMO protein was increased by the presence of NaCl in younger leaves but decreased in older leaves. The tissue localization of CMO and BADH proteins was analyzed by immunofluorescent labeling method using their primary antibodies and a fluorescein-conjugated secondary antibody. CMO and BADH proteins were constitutively co-localized in mesophyll and bundle sheath cells under both normal and saline conditions. A possible physiological function of GB in the salt tolerance of barley plants is discussed.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [14C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [14C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

Saline stress is one of the major factors limiting crop production in the world. Production of salt tolerant crops is, there- fore, a significant subject in agribiotechnology. We transformed a Japonica rice ( Oryza sativa cv. Nipponbare) and Indica rices (O. sativa cvs. Kasalath and BR5) with a gene encoding catalase, katE, derived from Escherichia coli which decomposes H2O2, one of reactive oxygen species produced by saline stress, and acts as a quencher of damage by H 2 O 2 . Rice plants were transformed using Agrobacterium tumefaciens EHA101 carrying pIG121/Hm/katE. The presence of katE gene in transgenic plants was confirmed by PCR, Southern blot and the expression of katE gene was detected by RT-PCR. Catalase activities of katE transgenic T1 plants are about 1.5 to 2.5 fold higher level than those of non transgenic plants. Transgenic plants (T 0 , T 1 ) of Nipponbare could grow even in 250mM NaCl solution for 14 days and seeds could be obtained when T1 transgenic plants were cultured throughout in 100mM NaCl solution. The transgenic plants of Kasalath and BR5 could grow in 100mM NaCl solution. Non-transformed plants of all cultivars did not grow in 50mM and higher concentrations of NaCl. Production of marker-free transgenic plants is required to get public acceptance. We used MAT Vector to produce marker-free salt tolerant Nipponbare. In MAT Vector System, selection markers are removed by recombinase and only katE remains. We succeeded to produce marker-free transgenic plants which could grow in 200mM NaCl solution. These findings reported here indicate the feasibility of producing salt tolerant transgenic plants to expand available lands for cultivation of crops on earth.

The accumulation of glycinebetaine (GB) is one of the adaptive strategies to adverse salt stress conditions. Although it has been demonstrated that barley plants accumulate GB in response to salt stress and various studies focused on GB synthesis were performed, its transport mechanism is still unclear. In this study, we identified a novel gene, HvProT2, encoding Hordeum vulgare GB/proline transporter from barley plants. Heterologous expression in yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of HvProT2 was highest for GB, intermediate for proline and lowest for γ-aminobutyric acid. Transient expression of fusions of HvProT2 and green fluorescent protein in onion epidermal cells revealed that HvProT2 is localized at the plasma membrane. Relative quantification of mRNA level of HvProT2 using semi-quantitative reverse transcription-polymerase chain reaction analysis showed that HvProT2 is constitutively expressed in both leaves and roots, and the expression level was higher in old leaves than young leaves and roots. Moreover, we found that HvProT2 was expressed in the mestome sheath and lateral root cap cells. We discussed the possible involvement of HvProT2 for salt stress tolerance.

Although rice (Oryza sativa L.) produces little glycine betaine (GB), it has two betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) gene homologs (OsBADH1 and OsBADH2). We found that OsBADH1 catalyzes the oxidation of acetaldehyde efficiently, while the activity of OsBADH2 is extremely low. The accumulation of OsBADH1 mRNA decreases following submergence treatment, but quickly recovers after re-aeration. We confirmed that OsBADH1 localizes in peroxisomes. In this paper, a possible physiological function of OsBADH1 in the oxidation of acetaldehyde produced by catalase in rice plant peroxisomes is discussed.

Glycinebetaine is a major compatible solute accumulated in response to salt stress in barley plants. In this study, we investigated the glycinebetaine content and tissue localization of mRNA of two betaine aldehyde dehydrogenase (BADH) genes, BBD1 and BBD2, and of BADH proteins in barley plants grown under control and saline conditions. Glycinebetaine was increased by salt treatment, and accumulated more in younger leaves than in older ones under both control and saline conditions. While BBD1 and BBD2 genes were constitutively expressed in mesophyll cells of leaves under both control and saline conditions, the signal of BBD2 transcripts increased strongly in vascular parenchyma cells in salt-stressed leaves. In roots under saline conditions, BBD1 transcripts were detected in epidermal cells, and BBD2 transcripts were detected in the pericycle. Moreover, BADH proteins were detected around the xylem vessels of leaves, and in the pericycle and epidermal cells of roots grown under a saline condition. These results suggested that glycinebetaine is synthesized in vascular tissues of leaves and the pericycle of roots in barley plants grown under salt stress.

Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (<50mM) of hydrogen peroxide by applying 300mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00h and starch at 15:00h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.

Ascorbate peroxidase (APX) plays an important role in oxidative stress metabolism in higher plants. To determine the role of APX in protection against excessive-zinc-induced oxidative stress, transgenic Arabidopsis plants constitutively overexpressing a peroxisomal ascorbate peroxidase gene (HvAPX1) from barley were analyzed. In this study, we found that transgenic plants were more tolerant to zinc stress than wild-type plants. Under zinc stress, the concentration of hydrogen peroxide and malondialdehyde accumulation were higher in wild-type plants than in transgenic plants. Therefore, the mechanism of zinc tolerance in transgenic plants may be due to reduced oxidative stress damage. Under zinc stress, the activities of APX were significantly higher in transgenic plants than in wild-type plants. We also found that the zinc accumulation in the shoots were much higher in transgenic plants than in wild-type plants under zinc stress. In addition, we found that compared with wild-type plants, transgenic plants were more tolerant to excessive cadmium stress and accumulated more cadmium in shoots. These results suggest that HvAPX1 plays an important role in zinc and cadmium tolerance, and might be a candidate gene for developing high-biomass tolerant plants for phytoremediation of zinc- and cadmium-polluted environments.

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley (Hordeum vulgare L.), we reported previously the existence of two BADH genes (BBD1 and BBD2) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K(m) of 18.9 microM and 19.9 mM, respectively. In addition, V(max)/K(m) with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of omega-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4-N-trimethylaminobutyraldehyde and 3-N-trimethylaminopropionaldehyde.

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Transgenic tobacco plants expressing a molecular chaperone DnaK from a halotolerant cyanobacterium Aphanothece halophytica show enhanced seed yields as well as enhanced tolerances for salt and heat stresses. High-temperature treatment during the reproductive stage decreased total dry weight of seeds in both transgenic and wild-type tobacco, but more severely in the wild-type. Transgenic tobacco plants exhibited higher activities of ascorbate peroxidase and catalase than wild-type plants. Similar results were obtained for salt stress during the reproductive stage. Transgenic rice plants expressing ApDnaK was also constructed. Transgenic rice plants exhibited the enhanced activities for Calvin-cycle enzymes, and showed faster growth and higher seed yield compared with the wild-type rice under normal growth conditions. Transgenic rice plants also showed enhanced tolerance for high temperature and salt stress compared with the wild-type rice. These results suggest a relation of increased folding activity with enhanced stress tolerance, increased seed yield, and total plant biomass.